Research: Protein phosphorylation by protein kinases is an important regulatory mechanism that influences every aspect of cellular life. The majority of phosphoproteins are intracellular; however, numerous extracellular proteins are also phosphorylated. In fact, the first evidence of protein phosphorylation was in 1883, when the secreted milk protein casein was shown to contain phosphate. In hindsight, this was the first indication for the existence of protein kinases. Many secreted proteins are phosphorylated by protein kinases present within the lumen of the Golgi. However, these enzymes are poorly characterized and their activities have been ascribed to the category of orphan enzymes (enzymes that have not been molecularly identified). One such kinase has been biochemically characterized from highly enriched Golgi fractions and named Golgi casein kinase (G-CK). The proteins known as casein kinases are in fact cytosolic and nuclear proteins and do not mediate physiological phosphorylation of casein because they are spatially restricted from the secretory apparatus and the extracellular space. The G-CK specifically recognizes the consensus Ser-x-(Glu/pSer) (where x is any amino acid and Glu/pSer can be Glu or phospho-Ser) and this motif is phosphorylated in some 75% of human plasma and cerebrospinal fluid phosphoproteins. The candidate was the first to identify the G-CK as Fam20C, and showed that it belongs to a novel family of atypical protein kinases that localize within the Golgi apparatus and are secreted. Fam20C phosphorylates casein and several hormones specifically at S-x-E/pS motifs in the lumen of the Golgi. Indeed, many hormones contain phosphate within this motif; however, in most cases, established functions for phosphorylation are unknown. Prior to the candidates' discovery that Fam20C was a protein kinase, it had been suggested to be a novel regulator of Fibroblast Growth Factor 23 (FGF23). Fam20C null mice develop hypophosphatemic rickets (HR) as a result of elevated secretion of FGF23, a bone- derived hormone implicated in the pathogenesis of many human diseases including disorders of mineral metabolism and chronic kidney disease. Hypothesis: The aims of this proposal were designed to test the hypothesis that the secreted protein kinase Fam20C regulates FGF23 by phosphorylation. The hypothesis was formulated based on three recent observations: 1) Fam20C is a protein kinase that phosphorylates secreted proteins at S-x-E/pS (Tagliabracci et. al., Science 2012). 2) Fam20C null mice develop hypophosphatemic rickets due to an increase in FGF23 (Wang et. al., PLoS Genet. 2012) and 3) Fam20C phosphorylates FGF23 at Ser180 within an important regulatory motif and at 3 highly conserved amino acids within the C terminus that may affect binding of the hormone to its cognate receptor (preliminary data, unpublished). Research Objective: The overall objective of this proposal is to determine the molecular mechanisms by which Fam20C regulates FGF23 processing and activity. Approach: The candidate will utilize a combination of molecular biology, biochemistry, cell biology, and mouse genetics to accomplish the research objective. The mentored phase (K99, Specific Aims 1 and 2) will consist of in vitro and cell based approaches to determine the molecular mechanism by which Fam20C regulates FGF23. The candidate will take advantage of the resources and personnel at UCSD, both of which are outstanding and well-suited for this phase of the award. The independent phase (Specific Aim 3) will employ mouse models to study the regulation of FGF23 by Fam20C. Significance: The experiments proposed in this application will answer fundamental questions regarding the pathogenesis of several disorders of mineral metabolism, including hereditary disorders of hypophosphatemic rickets; affecting ~1 in every 20,000 newborns. Furthermore, the results will have important implications for patients with chronic kidney disease, a growing public health epidemic that affects 26 million Americans. Training: The proposed research will be conducted within the department of Pharmacology at the University of California, San Diego (UCSD), under the mentorship of Dr. Jack E. Dixon. Dr. Dixon is a member of the National Academy of Sciences, USA, a Royal Society Fellow, and has led a distinguished scientific career. In addition, collaborations were established with Kenneth White, PhD, a leader in the field of metabolic bone diseases and Joachim H. Ix, MD, a noted nephrologist and epidemiologist. The candidate will undertake coursework through UCSD, participate in regular seminars, and present his findings at national scientific meetings. Overall, the training environment is excellent and the proposed studies are well designed and innovative. The hypothesis addresses a significant question that should yield influential findings in endocrinology, nephrology cardiology and basic biomedical research. The mentoring and research skills expected to develop over the course of the award will provide a strong foundation for the candidate to successfully transition to become a prominent independent investigator and leader in the field of secreted protein phosphorylation.